Plasmid autonomously replicable in coryneform bacteria

ABSTRACT

A plasmid isolatable from  Corynebacterium thermoaminogenes , which comprises a gene coding for a Rep protein having the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having homology of 90% or more to the amino acid sequence shown in SEQ ID NO: 8, and has a size of about 4.4 kb or about 6 kb, or a derivative thereof.

This application is a divisional of Ser. No. 09/636,458, filed Aug. 11,2000, now U.S. Pat. No. 6,905,819, which is hereby incorporated byreference. All documents cited herein, as well as the foreign prioritydocument, JP1999-228391, filed Aug. 12, 1999, are also herebyincorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a novel plasmid derived fromCorynebacterium thermoaminogenes. The plasmid of the present inventioncan be utilized for improving coryneform bacteria, which are used forproducing useful substances such as L-amino acids.

Amino acids, including L-glutamic acid and L-lysine, are produced byfermentative methods using the coryneform bacteria, which generallybelong to the genus Brevibacterium, Corynebacterium, or Microbacterium,or variant strains thereof (Amino Acid Fermentation, Gakkai ShuppanCenter, pp. 195-215, 1986).

In the industrial fermentative production of amino acids, besidesimproving the yield relative to saccharides, shortening the culturetime, improving the amino acid concentration, and so forth, increasingthe culture temperature is an important technical factor that increasesthe economical efficiency. That is, the culture is usually performed atan optimum fermentation temperature, which is 31.5° C. forCorynebacterium glutamicum. After the culture is started, heat isgenerated during the fermentation, and hence amino acid production ismarkedly reduced if this heat output is not removed. Therefore, coolingequipment is required in order to maintain the optimum temperature ofthe culture broth. On the other hand, if the culture temperature can beelevated, it is then possible to decrease the energy required forcooling and the cooling equipment can be reduced in size.

Among coryneform bacteria, Corynebacterium thermoaminogenes has beenisolated as a coryneform bacterium that can grow in higher temperatures(Japanese Patent Application Laid-open (Kokai) No. 63-240779). Whereasgrowth of Corynebacterium glutamicum is markedly suppressed at 40° C.,Corynebacterium thermoaminogenes can grow at a temperature of about 40°C. or higher, and is therefore suitable for high temperaturefermentation.

Currently, reliability of DNA recombination techniques is steadilyimproving in Escherichia coli and coryneform bacteria. To improvemicroorganisms using DNA recombinant techniques, plasmids derived frommicroorganisms belonging to other species, genus or broad host spectrumvectors are often used. However, plasmids native to the objectivemicroorganism are generally used. In particular, when the optimumculture temperature for the objective microorganism to be improved isdifferent from that of a microorganism of the same species or genus, itis preferable to use a plasmid native to the microorganism.

To date, plasmids derived from coryneform bacteria which have beenobtained are pAM330 from Brevibacterium lactofermentum ATCC13869(Japanese Patent Application Laid-open (Kokai) No. 58-67669), pBL1 fromBrevibacterium lactofermentum ATCC21798 (Santamaria. R. et al., J. Gen.Microbiol., 130, pp.2237-2246, 1984), pHM1519 from Corynebacteriumglutamicum ATCC13058 (Japanese Patent Application Laid-open (Kokai) No.58-77895), pCG1 from Corynebacterium glutamicum ATCC31808 (JapanesePatent Application Laid-open (Kokai) No. 57-134500) and pGA1 fromCorynebacterium glutamicum DSM58 (Japanese Patent Application Laid-open(Kokai) No. 9-2603011).

However, no plasmid native to Corynebacterium thermoaminogenes has beenobtained at present.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a plasmid which isuseful for improving a coryneform bacterium that can grow at an elevatedtemperature, Corynebacterium thermoaminogenes.

The inventors of the present invention found that Corynebacteriumthermoaminogenes AJ12340 (FERM BP-1539), AJ12308 (FERM BP-1540), AJ12309(FERM BP-1541) and AJ12310 (FERM BP-1542) each harbored a crypticplasmid native to each strain, and successfully isolated and identifiedeach plasmid. Thus, they accomplished the present invention.

That is, the present invention provides a plasmid isolatable fromCorynebacterium thermoaminogenes, which comprises a gene (rep gene)coding for a Rep protein which has the amino acid sequence shown in SEQID NO: 2, or an amino acid sequence which has homology of 90% or more tothe foregoing amino acid sequence, and has a size of about 4.4 kb orabout 6 kb, or a derivative thereof.

Examples of the aforementioned plasmids include a plasmid isolatablefrom Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539), AJ12308(FERM BP-1540) or AJ12310 (FERM BP-1542), which has a size of about 4.4kb and is depicted in the restriction map shown in FIG. 1, and a plasmidisolatable from Corynebacterium thermoaminogenes AJ12309 (FERM BP-1541),which has a size of about 6 kb and is depicted in the restriction mapshown in FIG. 2.

Specific examples of the aforementioned plasmid include a plasmid whichcomprises a gene coding for a Rep protein having the amino acid sequenceshown in SEQ ID NO: 2, 4 or 6, and a plasmid which comprises a genecoding for a Rep protein having the amino acid sequence shown in SEQ IDNO: 8.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a restriction map of the plasmids pYM1, pYM2 and pYM3 of thepresent invention.

FIG. 2 is a restriction map of the plasmid pYM4 of the presentinvention.

FIG. 3 shows construction of pYMFK.

FIG. 4 shows construction of pYMK.

FIG. 5 shows construction of pYMC.

FIG. 6 shows construction of pK1.

DETAILED DESCRIPTION OF THE INVENTION

The plasmid of the present invention can be isolated fromCorynebacterium thermoaminogenes AJ12340 (FERM BP-1539), AJ12308 (FERMBP-1540), AJ12309 (FERM BP-1541) or AJ12310 (FERM BP-1542) according toa usual method for preparing a plasmid, such as the alkali method (Textfor Bioengineering Experiments, Edited by the Society for Bioscience andBioengineering, Japan, p. 105, Baifukan, 1992). FERM BP-1539 wasdeposited at the National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology (postal code 305-8566, 1-3Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Mar. 13, 1987 andgiven an accession number of FERM P-9277, and was transferred to aninternational depository and deposited under the provisions of theBudapest Treaty on Oct. 27, 1987. FERM BP-1540, FERM BP-1541 and FERMBP-1542 were deposited at the aforementioned depository on Mar. 10, 1987and given accession numbers of FERM P-9244, FERM P-9245 and FERM P-9246,and were transferred to an international depository and deposited underthe provisions of the Budapest Treaty on Oct. 27, 1987.

The inventors of the present invention isolated and identified plasmidsnative to each of the aforementioned Corynebacterium thermoaminogenesAJ12308 (FERM BP-1540), AJ12310 (FERM BP-1542), AJ12340 (FERM BP-1539)and AJ12309 (FERM BP-1541), and designated them as pYM1, pYM2, pYM3 andpYM4, respectively. These plasmids exist as double-stranded circular DNAin a cell of Corynebacterium thermoaminogenes. The nucleotide sequenceof the rep gene contained in pYM1 is shown in SEQ ID NO: 1, thenucleotide sequence of the rep gene contained in pYM2 is shown in SEQ IDNO: 3, the nucleotide sequence of the rep gene contained in pYM3 isshown in SEQ ID NO: 5, and the nucleotide sequence of the rep genecontained in pYM4 is shown in SEQ ID NO: 7. The amino acid sequencesthat can be encoded by the rep genes contained in these plasmids areshown in SEQ ID NOS: 2, 4, 6 and 8. pYM1, pYM2 and pYM3 each have a sizeof about 4.4 kb. pYM4 has a size of about 6 kb.

The numbers and sizes of fragments that can be obtained when pYM1, pYM2and pYM3 are digested with typical restriction enzymes are shown inTable 1. The numbers and sizes of fragments that can be obtained whenpYM4 is digested with typical restriction enzymes are shown in Table 2.Further, a restriction map of pYM1, pYM2 and pYM3 is shown in FIG. 1,and a restriction map of pYM4 is shown in FIG. 2.

TABLE 1 Restriction Number of DNA fragment enzyme digestion site (kb)BglII 0 — BamHI 2 1.8, 2.6 BstPI 1 4.4 EcoRI 1 4.4 HincII 4 0.3, 0.5,2.0, 1.6 HindIII 0 — KpnI 0 — NaeI 2 0.1, 4.3 NcoI 1 4.4 NheI 2 1.8, 2.6PmaCI 1 4.4 SacI 0 — SalI 0 — SacII 3 0.1, 1.4, 2.9 SmaI 3 0.1, 1.8, 2.5SphI 0 — Tth111I 1 4.4 XbaI 0 —

TABLE 2 Restriction Number of DNA fragment enzyme digestion site (kb)BglII 1 6.0 BamHI 2 3.8, 2.2 BstPI 2 1.2, 4.8 EcoRI 1 6.0 HincII 4 0.3,0.4, 1.2, 1.7, 2.4 HindIII 0 — KpnI 0 — NaeI 2 0.1, 5.9 NcoI 3 0.2, 2.8,3.0 NheI 3 0.1, 2.3, 3.6 PmaCI 0 — SacI 0 — SalI 0 — SacII 5 0.1, 0.2,0.9, 1.8, 3.0 SmaI 2 0.1, 5.9 SphI 0 — Tth111I 0 — XbaI 0 —

Determination of the nucleotide sequence of the plasmids of the presentinvention revealed that pYM1, pYM2, and pYM3 each contain 4368 bp, 4369bp and 4369 bp, respectively, have substantially the same structure, andhave homology of 99.9% to one another on the nucleotide sequence level.Further, pYM4 contains 5967 bp and has extremely high homology to pYM1,pYM2 and pYM3 in the about 4.4 kb region, while pYM4 only has homologyof about 81% when compared as a whole.

The plasmids contain respective rep genes which have high homology toone another. Homology was compared for the amino acid sequences of theRep proteins encoded by the rep genes (SEQ ID NOS: 2, 4, 6 and 8) andthe amino acid sequences of the Rep proteins encoded by rep genes ofknown plasmids derived from coryneform bacteria. Homology of 99% or morewas observed among pYM1, pYM2 and pYM3, and homology of 81.91% wasobserved between pYM2 and pYM4. On the other hand, they showed nohomology to the known plasmid pAM330 of a coryneform bacterium, and theyshowed homology of 80% or less to pGA1 and pCG1. The results are shownin Table 3. Thus, the plasmid of the present invention and the knownplasmids of coryneform bacteria are distinguishable based on thehomology of the Rep protein.

The homology is calculated according to the method described in Takashi,K. and Gotoh, O., J. Biochem., 92, 1173-1177 (1984).

TABLE 3 Homology of amino acid sequences of Rep protein encoded byvarious plasmids PYM2 pYM4 pGA1 pCG1 PYM2 — 81.91% 68.01% 70.73% PYM4 —— 69.39% 70.23% PGA1 — — — 75.31% PCG1 — — —   —

Since the plasmid of the present invention can sufficiently replicate incells of coryneform bacteria, including Corynebacteriumthermoaminogenes, the genetic information of a foreign gene can beexpressed in a host microorganism by inserting the foreign gene at anysite in the plasmid, or the derivative thereof, and transforming thehost microorganism with the resulting recombinant plasmid.

Examples of coryneform bacteria are listed below.

Corynebacterium acetoacidophilum

Corynebacterium acetoglutamicum

Corynebacterium callunae

Corynebacterium glutamicum

Corynebacterium thermoaminogenes

Corynebacterium lilium (Corynebacterium glutamicum)

Corynebacterium melassecola

Brevibacterium divaricatum (Corynebacterium glutamicum)

Brevibacterium lactofermentum (Corynebacterium glutamicum)

Brevibacterium saccharolyticum

Brevibacterium immariophilum

Brevibacterium roseum

Brevibacterium flavum (Corynebacterium glutamicum)

Brevibacterium thiogenitalis

A “derivative” of the plasmid of the present invention means a plasmidcomposed of a part of the plasmid of the present invention, or theplasmid of present invention and another DNA sequence. The “part of aplasmid” means a part containing a region essential for autonomousreplication of the plasmid. The plasmid of the present invention canreplicate in a host microorganism even if a region other than the regionessential for the autonomous replication of the plasmid (replicationcontrol region), that is, the region other than the region containingthe replication origin and genes necessary for the replication, isdeleted. In addition, a plasmid having such a deletion will have asmaller size. Therefore, a plasmid having such a deletion is preferredfor use as a vector. Furthermore, if a marker gene, such as a drugresistance gene, is inserted into the plasmid of the present inventionor a part thereof, it becomes easy to detect transformants thanks to thephenotype of the marker gene in the transformants. Examples of such amarker gene that can be used in the host include chloramphenicolresistance gene, kanamycin resistance gene, streptomycin resistancegene, tetracycline resistence gene, trimethoprim resistance gene,erythromycin resistance gene, and so forth.

Furthermore, if the plasmid of the present invention is made as ashuttle vector, which is autonomously replicable in coryneform bacteriaand other bacteria such as Escherichia coli, by ligating the plasmid ofthe present invention or a part thereof with a plasmid autonomouslyreplicable in the other bacteria such as Escherichia coli or a partthereof containing a replication control region thereof, manipulationscan be performed using Escherichia coli, such as preparation of plasmidand preparation of recombinant plasmid containing a target gene.Examples of a plasmid autonomously replicable in Escherichia coliinclude, for example, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399,pHSG398, RSF110, pMW119, pMW118, pMW219, pMW218, and so forth.

Although pYM1, pYM2, pYM3 and pYM4 are characterized by the restrictionmaps shown in FIGS. 1 and 2, it is not necessarily required that theplasmid of present invention have these restriction maps, and anyrestriction site may be deleted as long as such deletion does not affectthe autonomous replication ability. Furthermore, the plasmid of thepresent invention may contain a restriction site that is not containedin pYM1, pYM2, pYM3 and pYM4.

The derivative of the plasmid as described above can be constructed inthe same manner as the conventionally known construction of cloningvectors, expression vectors and so forth. In order to construct thederivative, it is preferable to determine the nucleotide sequences ofpYM1, pYM2, pYM3 and pYM4. The nucleotide sequences can be determined byknown methods, such as the dideoxy method.

In order to insert a foreign gene into the plasmid or the derivativethereof of the present invention, it is convenient to insert it into arestriction site of the plasmid or the derivative thereof. A restrictionsite which is present as a single digestion site is preferred. In orderto insert a foreign gene, the plasmid and the source of the foreigngene, such as genomic DNA, can be partially or fully digested with oneor more restriction enzymes that provide the same cohesive ends, e.g.,the same restriction enzyme, and they can be ligated under suitableconditions. They may also be blunt-end ligated.

For the preparation of plasmid DNA, digestion and ligation of DNA,transformation and so forth, methods well-known to those skilled in theart may be employed. Such methods are described in Sambrook, J.,Fritsch, E. F., and Maniatis, T., “Molecular Cloning: A LaboratoryManual, Second Edition”, Cold Spring Harbor Laboratory Press (1989), andso forth.

According to the present invention, a novel plasmid derived fromCorynebacterium thermoaminogenes is provided as described above.

EXAMPLES

Hereinafter, the present invention will be explained in more detail withreference to the following examples.

Example 1

Isolation and Characterization of Plasmids from CorynebacteriumThermoaminogenes (FERM BP-1539, FERM BP-1540, FERM BP-1541, FERMBP-1542)

Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539), AJ12308 (FERMBP-1540), AJ12309 (FERM BP-1541) and AJ12310 (FERM BP-1542) werecultured for 12 hours in CM2B liquid medium (Bacto-trypton (Difco): 1%,Bacto-yeast-extract (Difco): 1%, NaCl: 0.5%, biotin: 10 μg/L), andplasmid DNA fractions were obtained by the alkali method (Text forBioengineering Experiments, Edited by the Society for Bioscience andBioengineering, Japan, p.105, Baifukan, 1992). When these fractions wereanalyzed by agarose gel electrophoresis (Sambrook, J., Fritsch, E. F.,and Maniatis, T., “Molecular Cloning: A Laboratory Manual, SecondEdition”, Cold Spring Harbor Laboratory Press (1989)), DNA bands weredetected for all of the fractions, and hence it was demonstrated thatthe aforementioned strains harbored plasmids. The plasmids prepared fromFERM BP-1540, FERM BP-1542 and FERM BP-1539 were designated as pYM1,pYM2 and pYM3, respectively. The plasmid prepared from FERM BP-1541 wasdesignated as pYM4. The plasmids pYM1, pYM2 and pYM3 each had a lengthof about 4.4 kb, and the plasmid pYM4 had a length of about 6.0 kb.

The plasmids pYM1, pYM2, pYM3 and pYM4 were digested with restrictionenzymes Bg1II, BamHI, BstPI, EcoRI, HincII, HindIII, KpnI, NaeI, NcoI,NheI, PmaCI, SacI, SacII, SalI, SmaI, SphI, Tth111I and XbaI (producedby Takara Co.), and the lengths of the produced DNA fragments weremeasured by agarose gel electrophoresis. The electrophoresis wasperformed at 100 V/cm and a constant voltage for several hours by usinga 0.8% agarose gel. λ phage DNA (Takara Shuzo) digested with arestriction enzyme HindIII was used as molecular weight markers. Theresults obtained for pYM1, pYM2 and pYM3 are shown in Table 1. Theresults obtained for pYM4 are shown in Table 2. The restriction map ofpYM1, pYM2 and pYM3 is shown in FIG. 1, and the restriction map of pYM4is shown in FIG. 2, which were prepared based on the above results.

The results of nucleotide sequencing of pYM1, pYM2, pYM3 and pYM4 by thedideoxy method are shown in SEQ ID NOS: 1, 3, 5 and 7 respectively.

Example 2

Construction of the Shuttle Vector pYMFK Containing the Km ResistanceGene derived from Streptococcus Faecalis

Regions necessary for efficient replication of pYM2 in coryneformbacteria include an AT-rich region upstream from rep and a region whichaffects copy number downstream from rep, besides the region coding forrep.

Therefore, in order to obtain a shuttle vector that can replicate incoryneform bacteria and E. coli without impairing the replicationability of pYM2, a region enabling autonomous replication in E. coli anda selection marker were inserted into sites in the vicinity of the BstPIsite of pYM2.

First, a vector having a drug resistance gene of S. faecalis wasconstructed. The kanamycin resistance gene of S. faecalis was amplifiedby PCR from a known plasmid containing that gene. The nucleotidesequence of the kanamycin resistance gene of S. faecalis has alreadybeen elucidated (Trieu-Cuot, P. and Courvalin, P., Gene, 23 (3),pp.331-341 (1983)). Based on this sequence, primers having thenucleotide sequences shown as SEQ ID NOS: 16 and 17 were synthesized,and PCR was performed using pDG783 (Anne-Marie Guerout-Fleury et al.,Gene, 167, pp.335-337 (1995)) as a template to amplify a DNA fragmentcontaining the kanamycin resistance gene and its promoter. The above DNAfragment was purified by using SUPREC02 produced by Takara Shuzo Co.,Ltd., completely digested with restriction enzymes HindIII and HincII,and blunt-ended. The blunt-ending was performed by Blunting Kit producedby Takara Shuzo Co., Ltd. This DNA fragment and an amplification productobtained by PCR with primers having the nucleotide sequences shown asSEQ ID NOS: 18 and 19, and pHSG399 (see S. Takeshita et al., Gene, 61,pp.63-74 (1987)) as a template, and purification and blunt-ending weremixed and ligated. The ligation reaction was performed by using DNALigation Kit ver.2 produced by Takara Shuzo Co., Ltd. Competent cells ofEscherichia coli JM109 (produced by Takara Shuzo Co., Ltd.) weretransformed with the ligated DNA, and cultured overnight in L medium (10g/L of Bacto trypton, 5 g/L of Bacto yeast extract, 5 g/L of NaCl, and15 g/L of agar, pH 7.2) containing 10 μg/ml of IPTG(isopropyl-β-D-thiogalactopyranoside), 40 μg/ml of X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 25 μg/ml of kanamycin.Then, the formed blue colonies were subjected to single colony isolationto obtain transformants.

Plasmids were prepared from the transformants using the alkaline method(Text for Bioengineering Experiments, Edited by the Society forBioscience and Bioengineering, Japan, p.105, Baifukan, 1992), andrestriction maps were prepared. A plasmid having a restriction mapequivalent to that shown at a lower position in FIG. 6 was designated aspK1. This plasmid is stably harbored in Escherichia coli, and impartskanamycin resistance to a host. Moreover, since it contains the lacZ'gene, it is suitable for use as a cloning vector.

Then, a region containing the replication origin was amplified byPyrobest-Taq (Takara Shuzo Co., Ltd.) using pYM2 extracted from C.thermoaminogenes AJ12310 (FERM BP-1542) as a template (The entirenucleotide sequence of pYM2 is shown in SEQ ID NO: 9.) and the followingprimers were prepared based on a sequence in pYM2 near the BstPI site:

S1: 5′-AAC CAG GGG GAG GGC GCG AGG C-3′ (SEQ ID NO: 10)

S3: 5′-TCT CGT AGG CTG CAT CCG AGG CGG GG-3′ (SEQ ID NO: 11)

The reaction conditions were 94° C. for 5 minutes, followed by a cycleof 98° C. for 20 seconds, and 68° C. for 4 minutes, which was repeatedfor 30 cycles, and 72° C. for 4 minutes. After the reaction, the mixturewas stored at 4° C.

The resulting amplified fragment was purified using MicroSpin TM S-400HR columns produced by Amersham Pharmacia Biotech Co., blunt-ended usingDNA Blunting Kit produced by Takara Shuzo Co., Ltd., and then ligated topK1, which had been treated with HincII using DNA Ligation Kit. ver. 2produced by Takara Shuzo Co., Ltd. Competent cells of Escherichia coliJM109 (produced by Takara Shuzo) were transformed with the ligated DNAto obtain transformant strains.

Plasmids were prepared from the transformant strains using the alkalimethod (Text for Bioengineering Experiments, Edited by the Society forBioscience and Bioengineering, Japan, p.105, Baifukan, 1992) andrestriction maps of the plasmids were prepared. A restriction mapequivalent to that shown at a lower position in FIG. 3 was designated aspYMFK. pYMFK had a size of about 7.0 kb, and was able to autonomouslyreplicate in E. coli and coryneform bacteria and impart Km resistance toa host.

Example 3

Construction of pYMK Containing Km Resistance Gene Derived from Tn903

A region containing the replication origin was amplified in the samemanner as in Example 2 by using pYM2 extracted from C. thermoaminogenesAJ12310 (FERM BP-1542) as a template and the following primers:

S1XbaI: 5′-GCT CTA GAG CAA CCA GGG GGA GGG CGC GAG GC-3′ (SEQ ID NO: 12)S3XbaI: 5′-GCT CTA GAG CTC TCG TAG GCT GCA TCG GAG GCG GGG-3′ (SEQ IDNO: 13)

The obtained amplified fragment was purified by using MicroSpin TM S-400HR columns produced by Amersham Pharmacia Biotech Co., digested with arestriction enzyme XbaI produced by Takara Shuzo Co., Ltd., and thenligated to a fragment obtained by fully digesting pHSG299 (Takara ShuzoCo., Ltd.) with XbaI by using DNA Ligation Kit. ver. 2 produced byTakara Shuzo Co., Ltd. Competent cells of Escherichia coli JM109(produced by Takara Shuzo) were transformed with the ligated DNA toobtain transformant strains.

Plasmids were prepared from the transformant strains using the alkalimethod (Text for Bioengineering Experiments, Edited by the Society forBioscience and Bioengineering, Japan, p.105, Baifikan, 1992) andrestriction maps of the plasmids were prepared. A restriction mapequivalent to that shown at a lower position in FIG. 4 was designated aspYMK. pYMK had a size of about 7.0 kb, and was able to autonomouslyreplicate in E. coli and coryneform bacteria and impart Km resistance toa host.

Example 4

Construction of Shuttle Vector pYMC Containing Cm Resistance GeneDerived from Tn9

A region containing the replication origin was amplified in the samemanner as in Example 2 by using pYM2 extracted from C. thermoaminogenesAJ12310 (FERM BP-1542) as a template and the following primers:

S1XbaI: 5′-GCT CTA GAG CAA CCA GGG GGA GGG CGC GAG GC-3′ (SEQ ID NO: 14)S3XbaI: 5′-GCT CTA GAG CTC TCG TAG GCT GCA TCG GAG GCG GGG-3′ (SEQ IDNO: 15)

The above DNA was purified by using MicroSpin TM S-400 HR columnsproduced by Amersham Pharmacia Biotech Co., digested with a restrictionenzyme XbaI produced by Takara Shuzo Co., Ltd., and then ligated to afragment obtained by treating pHSG399 (Takara Shuzo Co., Ltd.) with XbaIusing DNA Ligation Kit. ver. 2 produced by Takara Shuzo Co. Ltd.Competent cells of Escherichia coli JM109 (produced by Takara Shuzo)were transformed with the ligated DNA to obtain transformant strains.

Plasmids were prepared from the transformant strains using the alkalimethod (Text for Bioengineering Experiments, Edited by the Society forBioscience and Bioengineering, Japan, p. 105, Baifukan, 1992) andrestriction maps of the plasmids were prepared. One showing arestriction map equivalent to that shown at a lower position in FIG. 5was designated as pYMC. pYMC had a size of about 6.6 kb, and was able toautonomously replicate in E. coli and coryneform bacteria and impart Cmresistance to a host.

1. An isolated plasmid comprising a gene, said gene encoding apolypeptide having Rep protein activity and, said polypeptide comprisingan amino acid sequence that is at least 90% homologous to the amino acidsequence of SEQ ID NO:
 8. 2. The plasmid according to claim 1, whereinsaid polypeptide comprises an amino acid sequence that is at least 99%homologous to the amino acid sequence of SEQ ID NO:
 8. 3. The plasmidaccording to claim 1, wherein said polypeptide comprises the amino acidsequence of SEQ ID NO:
 8. 4. The plasmid according to claim 1, whereinsaid plasmid is isolated from Corynebacterium thermoaminogenes AJ12309.5. The plasmid according to claim 1, which has the restriction map shownin FIG.
 2. 6. The plasmid according to claim 1, wherein said gene isobtained from a plasmid having the restriction map shown in FIG.
 2. 7.The method of isolating the plasmid according to claim 1, comprising (A)culturing a Corynebacteriuin thermoaminogenes in a culture medium, (B)obtaining fractions by an alkali method, and (C) isolating said plasmid.8. The method according to claim 7, further comprising analyzing thefractions by agarose gel electrophoresis.
 9. An isolated polynucleotidecomprising a ucleic acid sequence that encodes a polypeptide having Repprotein activity and, said polypeptide comprising an amino acid sequencethat is at least 90% homologous to the amino acid sequence of SEQ ID NO.8.
 10. The polynucleotide according to claim 9, wherein said polypeptidecomprises an amino acid sequence that is at least 99% homologous to theamino acid sequence of SEQ ID NO:8.
 11. The polynucleotide according toclaim 9, wherein said polypeptide comprises the amino acid sequence ofSEQ ID NO:8.